Chimeric antigen receptors targeting HER2

ABSTRACT

Chimeric transmembrane immunoreceptors (CAR) which include an extracellular domain targeted to HER2, a transmembrane region, a costimulatory domain and an intracellular signaling domain are described.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a National Stage Application under 35 U.S.C. § 371and claims the benefit of International Application No.PCT/US2016/060724, filed Nov. 4, 2016, which claims priority to U.S.Application No. 62/251,052, filed Nov. 4, 2015. The disclosure of theforegoing application is hereby incorporated by reference in itsentirety.

BACKGROUND

Tumor-specific T cell based immunotherapies, including therapiesemploying engineered T cells, have been investigated for anti-tumortreatment. In some cases the T cells used in such therapies do notremain active in vivo for long enough periods. In some cases, thetumor-specificity of the T cells is relatively low, in part because ofthe heterogeneous nature of solid tumors and the potential foroff-target effects on non-cancerous cells when targeting self antigens.Therefore, there is a need in the art for tumor-specific cancertherapies with improved anti-tumor specificity and function.

Chimeric antigen receptors (CARs) are composed of an extracellular tumorrecognition/targeting domain, an extracellular linker/spacer, atransmembrane domain, and intracellular T cell-activating andco-stimulatory signaling domains. The design of therecognition/targeting domain is critical to avoiding deleteriousoff-target effects. The majority of CAR tumor targeting domains aresingle chain variable fragments (scFvs) derived from antibody sequencesthat exploit the specificity of antibody binding to particular antigens.There are also examples of CAR tumor targeting domains derived fromnormal receptor ligands, such as the IL-13 cytokine CAR that targetscells expressing the IL-13 receptor, IL13Rα2.

Adoptive T cell therapy (ACT) utilizing engineered T cells expressing aCAR has demonstrated robust and durable clinical efficacy in patientswith CD 19+ B-cell malignancies (Priceman et al. 2015 Curr Opin Oncol;Maus et al. 2014 Blood 123: 2625-2635. With early successes inhematologic diseases, broader application of this approach to solidtumors is now under intense investigation.

According to the National Cancer Institute Surveillance, Epidemiology,and End Results Program (SEER) data, there were an estimated 40,000deaths from breast cancer in 2014, primarily from metastatic disease.Approximately 25-30% of breast cancer patients carry an amplification ofthe HER2 gene, which confers a particularly poor prognosis. Even withthe advent of newer agents, including targeted therapies, there havebeen only modest improvements in overall mortality rates in Stage IVdisease. For example, in a randomized trial with HER2-positive breastcancer patients, the most promising treatment combination of twoHER2-targeted antibodies, trastuzumab and pertuzumab, plus docetaxelyields a median overall survival of 56.5 months and an extension ofprogression-free survival of only 6.3 months over trastuzumab anddocetaxel alone.

SUMMARY

Described herein are chimeric transmembrane immunoreceptors (chimericantigen receptors or “CARs”) which comprise an extracellular domain, atransmembrane region and an intracellular signaling domain. Theextracellular domain includes a scFv targeted to HER2 and, optionally, aspacer, comprising, for example, a portion of human Fe domain. Thetransmembrane portion includes, for example, a CD4 transmembrane domain,a CD8 transmembrane domain, a CD28 transmembrane domain, or a CD3transmembrane domain. The intracellular signaling domain includes thesignaling domain from the zeta chain of the human CD3 complex (CD3ξ) andone or more costimulatory domains, for example, a 4-IBB or a CD28costimulatory domain. The extracellular domain enables the CAR, whenexpressed on the surface of a T cell, to direct T cell activity to thosecells expressing HER2. Such cells include certain breast cancer cellsand certain brain cancer cells. The inclusion of a costimulatory domain,such as the 4-IBB (CD137) costimulatory domain in series with CD3ξ inthe intracellular region enables the T cell to receive co-stimulatorysignals. T cells, for example, patient-specific, autologous T cells canbe engineered to express the CARs described herein, and the engineeredcells can be expanded and used in ACT. Various T cell subsets, includingboth alpha beta T cells and gamma delta T cells, can be used. Inaddition, the CAR can be expressed in other immune cells such as NKcells. Where a patient is treated with an immune cell expressing a CARdescribed herein the cell can be an autologous T cell or an allogenic Tcell. In some cases the cells used are a cell population that includesboth CD4+ and CD8+ central memory T cells (T_(CM)), which are CD62L+,CCR7+, CD45RO+, and CD45RA−, or the cells used are a cell populationthat includes CD4+ and CD8+T_(CM) cells, stem central memory T cells andnaive T cells (i.e., a population of T_(CM/SCM/N) cells). A populationof T_(CM/SCM/N) cells are CD62L+, CCR7+ and include both CD45RA+ andCD45RO+ cells as well as both CD4+ cells and CD8+ cells. The use of suchcells can improve long-term persistence of the cells after adoptivetransfer compared to the use of other types of patient-specific T cells.

Described herein is a nucleic acid molecule encoding a CAR comprising:an scFv targeted to HER2 (e.g.,DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGSTSGGGSGGGSGGGGSSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS; SEQ ID NO: 1) or a variant thereof having1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); atransmembrane domain selected from: a CD4 transmembrane domain orvariant thereof having 1-5 (e.g., 1 or 2) amino acid modifications(e.g., substitutions), a CD8 transmembrane domain or variant thereofhaving 1-5 (e.g., 1 or 2) amino acid modifications (e.g.,substitutions), a CD28 transmembrane domain or a variant thereof having1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions), and aCD3ξ transmembrane domain or a variant thereof having 1-5 (e.g., 1 or 2)amino acid modifications (e.g., substitutions); a costimulatory domain(e.g., a CD28 co-stimulatory domain or a variant thereof having 1-5(e.g., 1 or 2) amino acid modifications (e.g., substitutions); or a4-1BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or2) amino acid modifications (e.g., substitutions); or both a CD28co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2)amino acid modifications (e.g., substitutions) and a 4-1BBco-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2)amino acid modifications (e.g., substitutions); and a CD3ξ signalingdomain or a variant thereof having 1-5 (e.g., 1 or 2) amino acidmodifications.

In various embodiments: the costimulatory domain is selected from thegroup consisting of: a CD28 costimulatory domain or a variant thereofhaving 1-5 (e.g., 1 or 2) amino acid modifications, a 4-1BBcostimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2)amino acid modifications and an OX40 costimulatory domain or a variantthereof having 1-5 (e.g., 1 or 2) amino acid modifications. In certainembodiments, a 4-1BB costimulatory domain or a variant thereof having1-5 (e.g., 1 or 2) amino acid modifications in present. In someembodiments there are two costimulatory domains, for example a CD28co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2)amino acid modifications (e.g., substitutions) and a 4-1BBco-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2)amino acid modifications (e.g., substitutions). In various embodimentsthe 1-5 (e.g., 1 or 2) amino acid modification are substitutions.

In some cases there is a short sequence of 1-6 amino acids (e.g. GGG)between the co-stimulatory domains and the CD3ξ signaling domain and/orbetween the two co-stimulatory domains.

Additional embodiment the CAR comprises: an scFv targeted to HER2; twodifferent costimulatory domains selected from the group consisting of: aCD28 costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2)amino acid modifications, a 4-1BB costimulatory domain or a variantthereof having 1-5 (e.g., 1 or 2) amino acid modifications and an OX40costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2)amino acid modifications; two different costimulatory domains selectedfrom the group consisting of: a CD28 costimulatory domain or a variantthereof having 1-2 amino acid modifications, a 4-1BB costimulatorydomain or a variant thereof having 1-2 amino acid modifications and anOX40 costimulatory domain or a variant thereof having 1-2 amino acidmodifications; a HER2 scFv or a variant thereof having 1-2 amino acidmodifications; a transmembrane domain selected from: a CD4 transmembranedomain or variant thereof having 1-2 amino acid modifications, a CD8transmembrane domain or variant thereof having 1-2 amino acidmodifications, a CD28 transmembrane domain or a variant thereof having1-2 amino acid modifications, and a CD3 transmembrane domain or avariant thereof having 1-2 amino acid modifications; a costimulatorydomain (e.g., a CD28 co-stimulatory domain or a variant thereof having1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); or a4-1BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or2) amino acid modifications (e.g., substitutions); or both a CD28co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2)amino acid modifications (e.g., substitutions) and a 4-1BBco-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2)amino acid modifications (e.g., substitutions); and CD3ξ signalingdomain of a variant thereof having 1-2 amino acid modifications; aspacer region located between the HER2 scFv or variant thereof and thetransmembrane domain (e.g., the spacer region comprises an amino acidsequence selected from the group consisting of SEQ ID NOs: 2-12 and 42(Table 3) or a variant thereof having 1-5 (e.g., 1 or 2) amino acidmodifications); the spacer comprises an IgG hinge region; the spacerregion comprises 1-150 amino acids; there is no spacer; the 4-1 BBsignaling domain comprises the amino acid sequence of SEQ ID NO:24 theCD3 signaling domain comprises the amino acid sequence of SEQ ID NO:21and a linker of 3 to 15 amino acids that is located between thecostimulatory domain and the CD3 signaling domain or variant thereof. Incertain embodiments where there are two costimulatory domains, one is a4-1BB costimulatory domain and the other a costimulatory domain selectedfrom: CD28 and CD28gg. In various embodiments the 1-5 (e.g., 1 or 2)amino acid modification are substitutions, e.g., conservativesubstitutions.

In some embodiments: nucleic acid molecule expresses a polypeptidecomprising an amino acid sequence selected from SEQ ID NOs: 26-41; thechimeric antigen receptor comprises an amino acid sequence selected fromSEQ ID NOs: 26-41.

Also disclosed is a population of human T cells transduced by a vectorcomprising an expression cassette encoding a chimeric antigen receptor,wherein chimeric antigen receptor comprises: an scFv targeted to HER2; atransmembrane domain selected from: a CD4 transmembrane domain orvariant thereof having 1-5 amino acid modifications (e.g., 1 or 2) aminoacid modifications (e.g., substitutions), a CD8 transmembrane domain orvariant thereof having 1-5 amino acid modifications (e.g., 1 or 2) aminoacid modifications (e.g., substitutions), a CD28 transmembrane domain ora variant thereof having 1-5 amino acid modifications (e.g., 1 or 2)amino acid modifications (e.g., substitutions), and a CD3ξ transmembranedomain or a variant thereof having 1-5 amino acid modifications (e.g., 1or 2) amino acid modifications (e.g., substitutions); a costimulatorydomain (e.g., a CD28 co-stimulatory domain or a variant thereof having1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); or a4-1 BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or2) amino acid modifications (e.g., substitutions); or both a CD28co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2)amino acid modifications (e.g., substitutions) and a 4-1BBco-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2)amino acid modifications (e.g., substitutions); and CD3ξ signalingdomain of a variant thereof having 1-5 amino acid modifications (e.g., 1or 2) amino acid modifications (e.g., substitutions). In variousembodiments: the population of human T cells comprise a vectorexpressing a chimeric antigen receptor comprising an amino acid sequenceselected from any of SEQ ID NOs: 26-41 or a variant thereof having 1-5amino acid modifications (e.g., 1 or 2) amino acid modifications (e.g.,substitutions); the population of human T cells comprises central memoryT cells (T_(CM) cells) e.g., at least 20%, 30%, 40%, 50% 60%, 70%, 80%of the cells are T_(CM) cells, or the population of T cells comprises acombination of central memory T cells, naive T cells and stem centralmemory cells (T_(CM/SCM/N) cells) e.g., at least 20%, 30%, 40%, 50% 60%,70%, 80% of the cells are T_(CM/SCM/N) cells. In either case, thepopulation of T cells includes both CD4+ cells and CD8+ cells (e.g., atleast 20% of the CD3+ T cells are CD4+ and at least 3% of the CD3+ Tcells are CD8+ and at least 70, 80 or 90% are either CD4+ or CD8+; atleast 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60% of the cells CD3+ cells areCD4+ and at least 4%, 5%, 8%, 10%, 20 of the CD3+ cells are CD8+ cells).

Also described is a method of treating cancer in a patient comprisingadministering a population of autologous or allogeneic human T cells(e.g., autologous or allogenic T cells comprising central memory T cells(T_(CM) cells) or a combination of central memory T cells, naive T cellsand stem central memory cells (i.e., the T cells are T_(CM/SCM/N) cells)at least 20%, 30%, 40%, 50% 60%, 70%, 80% of the cells are T_(CM/SCM/N)cells. In either case, the population of T cells includes both CD4+cells and CD8+ cells (e.g., at least 20% of the CD3+ T cells are CD4+and at least 3% of the CD3+ T cells are CD8+ and at least 70, 80 or 90%are either CD4+ or CD8+; at least 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%of the cells CD3+ cells are CD4+ and at least 4%, 5%, 8%, 10%, 20 of theCD3+ cells are CD8+ cells) transduced by a vector comprising anexpression cassette encoding a chimeric antigen receptor, whereinchimeric antigen receptor comprises an amino acid sequence selected fromSEQ ID NOs: 26-41 or a variant thereof having 1-5 (e.g., 1 or 2) aminoacid modifications (e.g., substitutions). In various embodiments: thecancer is brain cancer, e.g., an HER2-expressing brain cancer that is ametastasis from breast cancer; and the transduced human T cells whereprepared by a method comprising obtaining T cells from the patient,treating the T cells to isolate central memory T cells, and transducingat least a portion of the central memory cells to with a viral vectorcomprising an expression cassette encoding a chimeric antigen receptor,wherein chimeric antigen receptor comprises an amino acid sequenceselected from SEQ ID NOs: 26 or 27 or a variant thereof having 1-5(e.g., 1 or 2) amino acid modifications (e.g., substitutions). In somecases the CAR T cells are administered not directly the brain tumor, butare instead administered to the intraventricular space within the brainof the patient.

Also described is: a nucleic acid molecule encoding a polypeptidecomprising an amino acid sequence that is at least 95% identical to anamino acid sequence selected from SEQ ID NOs 26-41; a nucleic acidmolecule encoding an polypeptide comprising an amino acid sequence thatis identical to an amino acid sequence selected from SEQ ID NOs: 26-41except for the presence of no more than 5 amino acid substitutions,deletions or insertions; a nucleic acid molecule encoding an polypeptidecomprising an amino acid sequence that is identical to an amino acidsequence selected from SEQ ID NOs:26-41 except for the presence of nomore than 5 amino acid substitutions; and a nucleic acid moleculeencoding an polypeptide comprising an amino acid sequence that isidentical to an amino acid sequence selected from SEQ ID NOs:26-41except for the presence of no more than 2 amino acid substitutions.

This disclosure also includes nucleic acid molecules that encode any ofthe CARs described herein (e.g., vectors that include a nucleic acidsequence encoding one of the CARs) and isolated T lymphocytes thatexpress any of the CARs described herein.

The CAR described herein can include a spacer region located between theHER2 binding domain (e.g., a HER2 scFv) and the transmembrane domain. Avariety of different spacers can be used. Some of them include at leastportion of a human Fc region, for example a hinge portion of a human Fcregion or a CH3 domain or variants thereof. Table 1 below providesvarious spacers that can be used in the CARs described herein.

TABLE 1 Examples of Spacers Name Length Sequence a3   3 aa AAA linker 10 aa GGGSSGGGSG (SEQ ID NO: 2) IgG4 hinge   12 aaESKYGPPCPPCP (SEQ ID NO: 3) (S→P) (S228P) IgG4 hinge  12 aaESKYGPPCPSCP (SEQ ID NO: 4) IgG4 hinge   22 aa ESKYGPPCPPCPGGGSSGGGSG(S228P) +  (SEQ ID NO: 5) linker CD28 hinge  39 aaIEVMYPPPYLDNEKSNGTIIHVKGKHL CPSPLFPGPSKP (SEQ ID NO: 6) CD8 hinge- 48 aa AKPTTTPAPRPPTPAPTIASQPLSLRPEACR 48 aa PAAGGAVHTRGLDFACD(SEQ ID NO: 7) CD8 hinge-  45 aa TTTPAPRPPTPAPTIASQPLSLRPEACR 45 aaPAAGGAVHTRGLDFACD (SEQ ID NO: 8 IgG4 129 aa ESKYGPPCPPCPGGGSSGGGSGGQPR(HL-CH3) EPQVYTLPPSQEEMTKNQVSLTCLVK (includes GFYPSDIAVEWESNGQPENNYKTTPPS228P in  VLDSDGSFFL YSRLTVDKSRWQEGNV hinge) FSCSVMHEALHNHYTQKSLSLSLGKSEQ ID NO: 9) IgG4 229 aa ESKYGPPCPSCPAPEFEGGPSVFLFPPK (L235E, PKDTLMISRTPEVTCVVVDVSQEDPE N297Q) VQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCK VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL TCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV MHEALHNHYTQKSLSLSLGK (SEQ ID NO: 10) IgG4229 aa ESKYGPPCPPCPAPEFEGGPSVFLFPPK (S228P,  PKDTLMISRTPEVTCVVVDVSQEDPEL235E,  VQFNWYVDGVEVHQAKTKPREEQFQ N297Q) STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY TLPPSQEEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 11) IgG4 107 aaGQPREPQVYTLPPSQEEMTKNQVSLT (CH3) CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ EGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 12)HL  22 aa ESKYGPPCPPCPGGGSSGGGSG (SEQ ID NO: 42)

Some spacer regions include all or part of an immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4) hinge region, i.e., the sequence that falls betweenthe CHI and CH2 domains of an immunoglobulin, e.g., an IgG4 Fe hinge ora CD8 hinge. Some spacer regions include an immunoglobulin CH3 domain orboth a CH3 domain and a CH2 domain. The immunoglobulin derived sequencescan include one or more amino acid modifications, for example, 1, 2, 3,4 or 5 substitutions, e.g., substitutions that reduce off-targetbinding.

An “amino acid modification” refers to an amino acid substitution,insertion, and/or deletion in a protein or peptide sequence. An “aminoacid substitution” or “substitution” refers to replacement of an aminoacid at a particular position in a parent peptide or protein sequencewith another amino acid. A substitution can be made to change an aminoacid in the resulting protein in a non-conservative manner (i.e., bychanging the codon from an amino acid belonging to a grouping of aminoacids having a particular size or characteristic to an amino acidbelonging to another grouping) or in a conservative manner (i.e., bychanging the codon from an amino acid belonging to a grouping of aminoacids having a particular size or characteristic to an amino acidbelonging to the same grouping). Such a conservative change generallyleads to less change in the structure and function of the resultingprotein. The following are examples of various groupings of aminoacids: 1) Amino acids with nonpolar R groups: Alanine, Valine, Leucine,Isoleucine, Proline, Phenylalanine, Tryptophan, Methionine; 2) Aminoacids with uncharged polar R groups: Glycine, Serine, Threonine,Cysteine, Tyrosine, Asparagine, Glutamine; 3) Amino acids with chargedpolar R groups (negatively charged at pH 6.0): Aspartic acid, Glutamicacid; 4) Basic amino acids (positively charged at pH 6.0): Lysine,Arginine, Histidine (at pH 6.0). Another grouping may be those aminoacids with phenyl groups: Phenylalanine, Tryptophan, and Tyrosine.

In certain embodiments, the spacer is derived from an IgG 1, IgG2, IgG3,or IgG4 that includes one or more amino acid residues substituted withan amino acid residue different from that present in an unmodifiedspacer. The one or more substituted amino acid residues are selectedfrom, but not limited to one or more amino acid residues at positions220, 226, 228, 229, 230, 233, 234, 235, 234, 237, 238, 239, 243, 247,267, 268, 280, 290, 292, 297, 298, 299, 300, 305, 309, 218, 326, 330,331, 332, 333, 334, 336, 339, or a combination thereof. In thisnumbering scheme, described in greater detail below, the first aminoacid in the IgG4(L235E,N297Q) spacer in Table 1 is 219 and the firstamino acid in the IgG4(HL-CH3) spacer in Table 1 is 219 as is the firstamino acid in the IgG hinge sequence and the IgG4 hinge linker (HL)sequence in Table 1

In some embodiments, the modified spacer is derived from an IgG1, IgG2,IgG3, or IgG4 that includes, but is not limited to, one or more of thefollowing amino acid residue substitutions: C220S, C226S, S228P, C229S,P230S, E233P, V234A, L234V, L234F, L234A, L235A, L235E, G236A, G237A,P238S, S239D, F243L, P247I, S267E, H268Q, S280H, K290S, K290E, K290N,R292P, N297A, N297Q, S298A, S298G, S298D, S298V, T299A, Y300L, V305I,V309L, E318A, K326A, K326W, K326E, L328F, A330L, A330S, A331S, P331S,1332E, E333A, E333S, E333S, K334A, A339D, A339Q, P396L, or a combinationthereof.

In certain embodiments, the modified spacer is derived from IgG4 regionthat includes one or more amino acid residues substituted with an aminoacid residue different from that present in an unmodified region. Theone or more substituted amino acid residues are selected from, but notlimited to, one or more amino acid residues at positions 220, 226, 228,229, 230, 233, 234, 235, 234, 237, 238, 239, 243, 247, 267, 268, 280,290, 292, 297, 298, 299, 300, 305, 309, 218, 326, 330, 331, 332, 333,334, 336, 339, or a combination thereof.

In some embodiments, the modified spacer is derived from an IgG4 regionthat includes, but is not limited to, one or more of the following aminoacid residue substitutions: 220S, 226S, 228P, 229S, 230S, 233P, 234A,234V, 234F, 234A, 235A, 235E, 236A, 237A, 238S, 239D, 243L, 247I, 267E,268Q, 280H, 290S, 290E, 290N, 292P, 297A, 297Q, 298A, 298G, 298D, 298V,299A, 300L, 305I, 309L, 318A, 326A, 326W, 326E, 328F, 330L, 330S, 331S,331S, 332E, 333A, 333S, 333S, 334A, 339D, 339Q, 396L, or a combinationthereof, wherein the amino acid in the unmodified spacer is substitutedwith the above identified amino acids at the indicated position.

For amino acid positions in immunoglobulin discussed herein, numberingis according to the EU index or EU numbering scheme (Kabat et al. 1991Sequences of Proteins of Immunological Interest, 5th Ed., United StatesPublic Health Service, National Institutes of Health, Bethesda, herebyentirely incorporated by reference). The EU index or EU index as inKabat or EU numbering scheme refers to the numbering of the EU antibody(Edelman et al. 1969 Proc Natl Acad Sci USA 63:78-85).

A variety of transmembrane domains can be used in the Table 2 includesexamples of suitable transmembrane domains. Where a spacer domain ispresent, the transmembrane domain is located carboxy terminal to thespacer domain.

TABLE 2 Examples of Transmembrane Domains Acces- Name sion LengthSequence CD3z J04132.1 21 aa LCYLLDGILFIYGVILTALFL (SEQ ID NO: 13) CD28NM_ 27 aa FWVLVVVGGVLACYSLLVTVAFIIFWV 006139 (SEQ ID NO: 14) CD28 NM_28 aa MFWVLVVVGGVLACYSLLVTVAFIIFWV (M) 006139 (SEQ ID NO: 15) CD4 M3516022 aa MALIVLGGVAGLLLFIGLGIFF (SEQ ID NO: 16) CD8tm NM_ 21 aaIYIWAPLAGTCGVLLLSLVIT 001768 (SEQ ID NO: 17) CD8tm2 NM_ 23 aaIYIWAPLAGTCGVLLLSLVITLY 001768 (SEQ ID NO: 18) CD8tm3 NM_ 24 aaIYIWAPLAGTCGVLLLSLVITLYC 001768 (SEQ ID NO: 19) 41BB NM_ 27 aaIISFFLALTSTALLFLLFF LTLRFSVV 001561 (SEQ ID NO: 20)

Many of the CAR described herein include one or more (e.g., two)costimulatory domains. The costimulatory domain(s) are located betweenthe transmembrane domain and the CD3ξ signaling domain. Table 3 includesexamples of suitable costimulatory domains together with the sequence ofthe CD3ξ signaling domain.

TABLE 3 CD3 Domain and Examples of Costimulatory Domains Acces- Namesion Length Sequence CD3ξ 104132.1 113 aa RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEA YSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR (SEQ ID NO: 21) CD28 NM   42 aaRSKRSRLLHSDYMNMTPRRPGPTRKHYQ 006139 PYAPPRDFAAYRS (SEQ ID NO: 22)CD28gg* NM   42 aa RSKRSRGGQHSDYMNMTPRRPGPTRKHY 006139 QPYAPPRDFAAYRS(SEQ ID NO: 23) 41BB NM   42 aa KRGRKKLL YIFKQPFMRPVQTTQEEDG 001561CSCRFPEEEEGGCEL (SEQ ID NO: 24) OX40  42 aa ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO: 25)

DESCRIPTION OF DRAWINGS

FIG. 1 depicts the amino acid sequence of Her2scFv-IgG4(S228P,L235E,N297Q)-CD28tm-CD28gg-Zeta-T2A-CD19t (SEQ ID NO: 26). The variousdomains are listed in order below the sequence and are indicated byalternating underlining and non-underlining. The mature CAR sequence(SEQ ID NO: 34) does not include the GMCSFRa signal peptide, theT2A skipsequence or truncated CD19. Also called HER2(EQ)8ξ in FIGS. 3A-8E andthroughout the specification.

FIG. 2 depicts the amino acid sequence ofHer2scFv-IgG4(S228P,L235E,N297Q)-CD8tm-41BB-Zeta-T2A-CD19t (SEQ IDNO:27). The various domains are listed in order below the sequence andare indicated by alternating underlining and non-underlining. The matureCAR sequence (SEQ ID NO: 35) does not include the GMCSFRa signalpeptide, theT2A skip sequence or truncated CD19. Also called HER2(EQ)BBξin FIGS. 3A-8E and throughout the specification.

FIGS. 3A-D depict HER2-specific CAR constructs and CAR T cell expansiondata.

FIGS. 4A-D depict in vitro characterization of HER2-CAR T cells againstbreast cancer cell lines.

FIGS. 5A-5F depict the result of studies on the in vitro tumor activityof HER2-CAR T cells.

FIGS. 6A-6I depict the result of studies on the in vivo anti-tumorefficacy of local intratumorally-delivered HER2-CAR T cells.

FIGS. 7A-7D depict the results of studies on local delivery of HER2-CART cells in human orthotopic BBM xenograft models.

FIGS. 8A-8D depict the results of studies on intraventrical delivery ofHER2-CAR T cells.

FIG. 9 depicts the amino acid sequence ofHer2scFv-CD8hinge-CD8tm-41BB-Zeta-T2A-CD19t (SEQ ID NO: 28). The variousdomains are listed in order below the sequence and are indicated byalternating underlining and non-underlining. The mature CAR sequence(SEQ ID NO: 36) does not include the GMCSFRa signal peptide, theT2A skipsequence or truncated CD 19.

FIG. 10 depicts the amino acid sequence ofHer2scFv-IgG4hinge(S228P)-linker-CD8tm-41BB-Zeta-T2A-CD19t. (SEQ IDNO:29) The various domains are listed in order below the sequence andare indicated by alternating underlining and non-underlining. The matureCAR sequence (SEQ ID NO: 37) does not include the GMCSFRa signalpeptide, theT2A skip sequence or truncated CD 19.

FIG. 11 depicts the amino acid sequence ofHer2scFv-IgG4(S228P)-Linker-IgG4 CH3-CD8tm-41BB-Zeta-T2A-CD19t (SEQ IDNO: 30). The various domains are listed in order below the sequence andare indicated by alternating underlining and non-underlining. The matureCAR sequence (SEQ ID NO: 38) does not include the GMCSFRa signalpeptide, theT2A skip sequence or truncated CD19.

FIG. 12 depicts the amino acid sequence ofHer2scFv-IgG4(S228P)-Linker-IgG4 CH3-CD28tm-CD28gg-Zeta-T2A-CD19t (SEQID NO: 31). The various domains are listed in order below the sequenceand are indicated by alternating underlining and non-underlining. Themature CAR sequence (SEQ ID NO: 39) does not include the GMCSFRa signalpeptide, theT2A skip sequence or truncated CD19.

FIG. 13 depicts the amino acid sequence ofHer2scFv-Linker-CD28tm-CD28gg-Zeta-T2A-CD19t (SEQ ID NO: 32). Thevarious domains are listed in order below the sequence and are indicatedby alternating underlining and non-underlining. The mature CAR sequence(SEQ ID NO: 40) does not include the GMCSFRa signal peptide, theT2A skipsequence or truncated CD19.

FIG. 14 depicts the amino acid sequence ofHer2scFv-Linker-CD8tm-41BB-Zeta-T2A-CD19t. (SEQ ID NO: 33). The variousdomains are listed in order below the sequence and are indicated byalternating underlining and non-underlining. The mature CAR sequence(SEQ ID NO: 41) does not include the GMCSFRa signal peptide, theT2A skipsequence or truncated CD19.

FIGS. 15A-15C depict the results of studies characterizing certainadditional CAR with various spacers. The IgG3(EQ) is in FIG. 2; DeltaCh2is in FIG. 11; CD8h is in FIG. 9; HL is in FIG. 10; and L is in FIG. 14.

FIGS. 16A-16C show the results of studies examining CD107a and INF gammaproduced when TCM expressing the varios CAAR are exposed to cells notexpressing HER2 (MDA-MB-468), low HER2 (231BR), low HER2 (231BRHER2LO)or high HER2 (231BRHER2HI).

FIGS. 17A-17D show the results of studies examining PD-1 production andtumor cell killing ins various cell lines with the CAR of FIG. 2(HER2(EQ)BBξ or FIG. 14 (HER2(L)BBξ).

FIGS. 18A-18B show the results of studies examining CD107a and INF gammaproduced when TCM expressing the varios CAAR are exposed to cells notexpressing HER2 (MDA-MB-468), low HER2 (231BR), low HER2 (231BRHER2LO)or high HER2 (231BRHER2HI).

DETAILED DESCRIPTION

Described below is the structure, construction and characterization ofvarious chimeric antigen receptors targeting HER2 and useful in treatingHER2-expressing breast cancer as well as breast to brain metastasis.Importantly, the CAR described herein can be used in ACT to treat HER2expressing tumors in the brain by intraventricular or intratumoraldelivery.

A chimeric antigen (CAR) is a recombinant biomolecule that contains, ata minimum, an extracellular recognition domain, a transmembrane region,and an intracellular signaling domain. The term “antigen,” therefore, isnot limited to molecules that bind antibodies, but to any molecule thatcan bind specifically to a target. For example, a CAR can include aligand that specifically binds a cell surface receptor. Theextracellular recognition domain (also referred to as the extracellulardomain or simply by the recognition element which it contains) comprisesa recognition element that specifically binds to a molecule present onthe cell surface of a target cell. The transmembrane region anchors theCAR in the membrane. The intracellular signaling domain comprises thesignaling domain from the zeta chain of the human CD3 complex andoptionally comprises one or more costimulatory signaling domains. CARscan both to bind antigen and transduce T cell activation, independent ofMHC restriction. Thus, CARs are “universal” immunoreceptors which cantreat a population of patients with antigen-positive tumors irrespectiveof their HLA genotype. Adoptive immunotherapy using T lymphocytes thatexpress a tumor-specific CAR can be a powerful therapeutic strategy forthe treatment of cancer.

In some cases the CAR described herein can be produced using a vector inwhich the CAR open reading frame is followed by a T2A ribosome skipsequence and a truncated CD19 (CD19t), which lacks the cytoplasmicsignaling tail (truncated at amino acid 323). In this arrangement,co-expression of CD19t provides an inert, non-immunogenic surface markerthat allows for accurate measurement of gene modified cells, and enablespositive selection of gene-modified cells, as well as efficient celltracking and/or imaging of the therapeutic T cells in vivo followingadoptive transfer. Co-expression of CD 19t provides a marker forimmunological targeting of the transduced cells in vivo using clinicallyavailable antibodies and/or immunotoxin reagents to selectively deletethe therapeutic cells, and thereby functioning as a suicide switch.

The CAR described herein can be produced by any means known in the art,though preferably it is produced using recombinant DNA techniques.Nucleic acids encoding the several regions of the chimeric receptor canbe prepared and assembled into a complete coding sequence by standardtechniques of molecular cloning known in the art (genomic libraryscreening, PCR, primer-assisted ligation, site-directed mutagenesis,etc.) as is convenient. The resulting coding region is preferablyinserted into an expression vector and used to transform a suitableexpression host cell line, preferably a T lymphocyte cell line, and mostpreferably an autologous T lymphocyte cell line.

Various T cell subsets isolated from the patient, including unselectedPBMC or enriched CD3 T cells or enriched CD3 or memory T cell subsets orT_(CM) or T_(CM/SCM/N) can be transduced with a vector for CARexpression. Central memory T cells are one useful T cell subset. Centralmemory T cell can be isolated from peripheral blood mononuclear cells(PBMC) by enriching for CD45RO+/CD62L+ cells, using, for example, theCliniMACS® device to immunomagnetically select cells expressing thedesired receptors. The cells enriched for central memory T cells can beactivated with anti-CD3/CD28, transduced with, for example, a SINlentiviral vector that directs the expression of the CAR as well as atruncated human CD19 (CD19t), a non-immunogenic surface marker for bothin vivo detection and potential ex vivo selection. Theactivated/genetically modified central memory T cells can be expanded invitro with IL-2/IL-15 and then cryopreserved.

Example 1: Structure of Two HER2-CAR

One CAR comprising a HER2 scFv described herein is referred to asHer2scFv-IgG4(L235E, N297Q)-CD28tm-CD28gg-Zeta-T2A-CD19t. This CARincludes a variety of important features including: a scFv targeted toHER2; an IgG4 Fe region that is mutated at two sites within the CH2region (L235E; N297Q) in a manner that reduces binding by Fe receptors(FcRs); a CD28 transmembrane domain, a CD28 co-stimulatory domain, andCD3ξ activation domain. FIG. 1 presents the amino acid sequence of thisCAR, including the sequence of the truncated CDI9 sequence used formonitoring CAR expression and the T2A ribosomal skip sequence thatallows the CAR to be produced without fusion of the truncated CDI9sequence. As shown in FIG. 2, the immature CAR includes: GMCSFR signalpeptide, HER2 scFv, IgG4 that acts as a spacer, a CD8 transmembranedomain, a 4-IBB co-stimulatory domain that includes a LL to GG sequencealteration, a three Gly sequence, CD3 Zeta stimulatory domain. Thetranscript also encodes a T2A ribosomal sequence and a truncated CDI9sequence that are not part of the CAR protein sequence. The mature CARis identical to the immature CAR, but lacks the GMCSF signal peptide.

Example 2: Construction and Structure of epHIV7 Used for Expression ofHER2-Specific CAR T Cells

The epHIV7 vector is a vector that can used for expression of theHER2-specific CAR. Is was produced from pHIV7 vector. Importantly, thisvector uses the human EFI promoter to drive expression of the CAR. Boththe 5′ and 3′ sequences of the vector were derived from pv653RSN aspreviously derived from the HXBc2 provirus. The polypurine tract DNAflap sequences (cPPT) were derived from HIV-I strain pNL4-3 from the NIHAIDS Reagent Repository. The woodchuck post-transcriptional regulatoryelement (WPRE) sequence was previously described.

Construction of pHIV7 was carried out as follows. Briefly, pv653RSN,containing 653 bp from gag-pol plus 5′ and 3′ long-terminal repeats(LTRs) with an intervening SL3-neomycin phosphotransferase gene (Neo),was subcloned into pBluescript, as follows: In Step I, the sequencesfrom 5′ LTR to rev-responsive element (RRE) made p5 ‘HIV-I 5I, and thenthe 5′ LTR was modified by removing sequences upstream of the TATA box,and ligated first to a CMV enhancer and then to the SV40 origin ofreplication (p5′HIV-2). In Step 2, after cloning the 3′ LTR intopBluescript to make p3′HIV-1, a 400-bp deletion in the 3′ LTRenhancer/promoter was made to remove cis-regulatory elements in HIV U3and form p3′HIV-2. In Step 3, fragments isolated from the p5′HIV-3 andp3′HIV-2 were ligated to make pHIV-3. In Step 4, the p3′HIV-2 wasfurther modified by removing extra upstream HIV sequences to generatep3′HIV-3 and a 600-bp BamHI-SalI fragment containing WPRE was added top3′HIV-3 to make the p3′HIV-4. In Step 5, the pHIV-3 RRE was reduced insize by PCR and ligated to a 5′ fragment from pHIV-3 (not shown) and tothe p3′HIV-4, to make pHIV-6. In Step 6, a 190-bp BglII-BamHI fragmentcontaining the cPPT DNA flap sequence from HIV-I pNL4-3 (55) wasamplified from pNL4-3 and placed between the RRE and the WPRE sequencesin pHIV 6 to make pHIV-7. This parent plasmid pHIV7-GFP (GFP, greenfluorescent protein) was used to package the parent vector using afour-plasmid system.

A packaging signal, psi (ψ), is required for efficient packaging ofviral genome into the vector. The RRE and WPRE enhance the RNAtranscript transport and expression of the transgene. The flap sequence,in combination with WPRE, has been demonstrated to enhance thetransduction efficiency of lentiviral vector in mammalian cells.

The helper functions, required for production of the viral vector), aredivided into three separate plasmids to reduce the probability ofgeneration of replication competent lentivirus via recombination: 1)pCgp encodes the gag/pol protein required for viral vector assembly; 2)pCMV-Rev2 encodes the Rev protein, which acts on the RRE sequence toassist in the transportation of the viral genome for efficientpackaging; and 3) pCMV-G encodes the glycoprotein of thevesiculo-stomatitis virus (VSV), which is required for infectivity ofthe viral vector.

There is minimal DNA sequence homology between the pHIV7 encoded vectorgenome and the helper plasmids. The regions of homology include apackaging signal region of approximately 600 nucleotides, located in thegag/pol sequence of the pCgp helper plasmid; a CMV promoter sequence inall three helper plasmids; and a RRE sequence in the helper plasmidpCgp. It is highly improbable that replication competent recombinantvirus could be generated due to the homology in these regions, as itwould require multiple recombination events. Additionally, any resultingrecombinants would be missing the functional LTR and tat sequencesrequired for lentiviral replication.

The CMV promoter was replaced by the EFlα-HTLV promoter (EFIp), and thenew plasmid was named epHIV7. The EFlp has 563 bp and was introducedinto epHIV7 using NruI and NheI, after the CMV promoter was excised.

The lentiviral genome, excluding gag/pol and rev that are necessary forthe pathogenicity of the wild-type virus and are required for productiveinfection of target cells, has been removed from this system. Inaddition, the vector construct does not contain an intact 3′LTRpromoter, so the resulting expressed and reverse transcribed DNAproviral genome in targeted cells will have inactive LTRs. As a resultof this design, no HIV-I derived sequences will be transcribed from theprovirus and only the therapeutic sequences will be expressed from theirrespective promoters. The removal of the LTR promoter activity in theSIN vector is expected to significantly reduce the possibility ofunintentional activation of host genes.

Example 3: Production of Vectors for Transduction of Patient T Cells

Vectors for transduction of patient T cells can be prepared as follows.For each plasmid the plasmid expressing the CAR and, optionally, amarker such as truncated CD19; 2) pCgp; 3) pCMV-G; and 4) pCMV-Rev2), aseed bank is generated, which is used to inoculate the fermenter toproduce sufficient quantities of plasmid DNA. The plasmid DNA is testedfor identity, sterility and endotoxin prior to its use in producinglentiviral vector.

Briefly, cells are expanded from the 293T working cell (WCB), which hasbeen tested to confirm sterility and the absence of viral contamination.A vial of 293T cells from the 293T WCB is thawed. Cells are grown andexpanded until sufficient numbers of cells exists to plate anappropriate number of 10 layer cell factories (CFs) for vectorproduction and cell train maintenance. A single train of cells can beused for production.

The lentiviral vector is produced in sub-batches of up to 10 CFs. Twosub-batches can be produced in the same week leading to the productionof approximately 20 L of lentiviral supernatant/week. The materialproduced from all sub-batches is pooled during the downstream processingphase, in order to produce one lot of product. 293T cells are plated inCFs in 293T medium (DMEM with 10% FBS). Factories are placed in a 37° C.incubator and horizontally leveled in order to get an even distributionof the cells on all the layers of the CF. Two days later, cells aretransfected with the four lentiviral plasmids described above using theCaPQ4 method, which involves a mixture of Tris:EDTA, 2M CaCh, 2×HBS, andthe four DNA plasmids. Day 3 after transfection, the supernatantcontaining secreted lentiviral vectors is collected, purified andconcentrated. After the supernatant is removed from the CFs,End-of-Production Cells are collected from each CF. Cells aretrypsinized from each factory and collected by centrifugation. Cells areresuspended in freezing medium and cryopreserved. These cells are laterused for replication-competent lentivirus (RCL) testing.

To purify and formulate vectors crude, supernatant is clarified bymembrane filtration to remove the cell debris. The host cell DNA andresidual plasmid DNA are degraded by endonuclease digestion(Benzonase®). The viral supernatant is clarified of cellular debrisusing a 0.45 μm filter. The clarified supernatant is collected into apre-weighed container into which the Benzonase® is added (finalconcentration 50 U/mL). The endonuclease digestion for residual plasmidDNA and host genomic DNA is performed at 37° C. for 6 h. The initialtangential flow ultrafiltration (TFF) concentration of theendonuclease-treated supernatant is used to remove residual lowmolecular weight components from the crude supernatant, whileconcentrating the virus ˜20 fold. The clarified endonuclease-treatedviral supernatant is circulated through a hollow fiber cartridge with aNMWCO of 500 kD at a flow rate designed to maintain the shear rate at˜4,000 sec⁻¹ or less, while maximizing the flux rate. Diafiltration ofthe nuclease-treated supernatant is initiated during the concentrationprocess to sustain the cartridge performance. An 80% permeatereplacement rate is established, using 4% lactose in PBS as thediafiltration buffer. The viral supernatant is brought to the targetvolume, representing a 20-fold concentration of the crude supernatant,and the diafiltration is continued for 4 additional exchange volumes,with the permeate replacement rate at 100%.

Further concentration of the viral product is accomplished by using ahigh speed centrifugation technique. Each sub-batch of the lentivirus ispelleted using a Sorvall RC-26 plus centrifuge at 6000 RPM (6,088 RCF)at 6° C. for 16-20 h. The viral pellet from each sub-batch is thenreconstituted in a 50 mL volume with 4% lactose in PBS. Thereconstituted pellet in this buffer represents the final formulation forthe virus preparation. The entire vector concentration process resultsin a 200-fold volume reduction, approximately. Following the completionof all of the sub-batches, the material is then placed at −80° C., whilesamples from each sub-batch are tested for sterility. Followingconfirmation of sample sterility, the sub-batches are rapidly thawed at37° C. with frequent agitation. The material is then pooled and manuallyaliquoted in the Class II Type A/B3 biosafety cabinet. A fillconfiguration of 1 mL of the concentrated lentivirus in sterile USPclass 6, externally threaded O-ring cryovials is used.

To ensure the purity of the lentiviral vector preparation, it is testedfor residual host DNA contaminants, and the transfer of residual hostand plasmid DNA. Among other tests, vector identity is evaluated byRT-PCR to ensure that the correct vector is present.

Example 4: Preparation of T Cells Suitable for Use in ACT

If T_(CM) are to be used to express the CAR, suitable patient cells canbe prepared as follows. First, T lymphocytes are obtained from a patientby leukopheresis, and the appropriate allogenic or autologous T cellsubset, for example, Central Memory T cells (T_(CM)), are geneticallyaltered to express the CAR, then administered back to the patient by anyclinically acceptable means, to achieve anti-cancer therapy.

Suitable TcM can be generated as follow. Apheresis products obtainedfrom consented research participants are ficolled, washed and incubatedovernight. Cells are then depleted of monocyte, regulatory T cell andnaïve T cell populations using GMP grade anti-CD14, anti-CD25 andanti-CD45RA reagents (Miltenyi Biotec) and the CliniMACS™ separationdevice. Following depletion, negative fraction cells are enriched forCD62L+ T_(CM) cells using DREG56-biotin (COH clinical grade) andanti-biotin microbeads (Miltenyi Biotec) on the CliniMACS™ separationdevice.

Following enrichment, T_(CM) cells are formulated in complete X-Vivo15plus 50 IU/mL IL-2 and 0.5 ng/mL IL-15 and transferred to a Teflon cellculture bag, where they are stimulated with Dynal ClinEx™ Vivo CD3/CD28beads. Up to five days after stimulation, cells are transduced withlentiviral vector expressing the desired CAR at a multiplicity ofinfection (MOI) of 1.0 to 0.3. Cultures are maintained for up to 42 dayswith addition of complete X-Vivo15 and IL-2 and IL-15 cytokine asrequired for cell expansion (keeping cell density between 3×10⁵ and2×10⁶ viable cells/mL, and cytokine supplementation every Monday,Wednesday and Friday of culture). Cells typically expand toapproximately 10⁹ cells under these conditions within 21 days. At theend of the culture period cells are harvested, washed twice andformulated in clinical grade cryopreservation medium (Cryostore CS5,BioLife Solutions).

On the day(s) of T cell infusion, the cryopreserved and released productis thawed, washed and formulated for re-infusion. The cryopreservedvials containing the released cell product are removed from liquidnitrogen storage, thawed, cooled and washed with a PBS/2% human serumalbumin (HSA) Wash Buffer. After centrifugation, the supernatant isremoved and the cells resuspended in a Preservative-Free Normal Saline(PFNS)/2% HSA infusion diluent. Samples are removed for quality controltesting.

Example 5: Expression of CAR Targeted to HER2

FIG. 3A is a schematic diagram of two the HER2-specific CAR constructsdepicted in FIG. 1 and FIG. 2. In HER2(EQ)28ξ the scFv is tethered tothe membrane by a modified IgG4 Fc linker (double mutant, L235E; N297Q),containing a CD28 transmembrane domain, an intracellular CD28co-stimulatory domain and a cytolytic CD3ξ domain. The T2A skip sequenceseparates the CAR from a truncated CD19 (CD19t) protein employed forcell tracking. HER2(EQ)BBξ is similar except that the costimulatorydomain is 4-1BB rather than CD28 and the transmembrane domain is a CD8transmembrane domain rather than a CD28 transmembrane domain. Humancentral memory (TCM) cells were transfected with a lentiviral vectorexpressing either HER2(EQ)28ξ or HER2(EQ)BBξ. FIG. 3B depictsrepresentative FACS data of human TCM surface phenotype. FIG. 3C depictsthe results of assays for CD 19 and Protein L expression in TCMtransfected with a lentiviral vector expressing either HER2(EQ)28ξ orHER2(EQ)BBξ. As can be seen from these results, transfection efficiencyas assessed by CD19 expression was similar for both CAR. However,Protein L expression was lower for HER2(EQ)BBξ than for HER2(EQ)28ξsuggesting that the HER2(EQ)BBξ CAR is less stable that the HER2(EQ)BBξ.Analysis of cell expansion (FIG. 3D) shows that neither CAR interfereswith T cell expansion.

Example 6: In Vitro Characterization of HER2-CAR T Cells Against VariousBreast Cancer Cell Lines

A variety of breast cancer cell lines, including, HER2-negative lines(LCL lymphoma, MDA-MB-468, U87 glioma), low-HER2 expressing lines(MDA-MB-361, 231BR) and high-HER2 expressing lines (SKBR3, BT474, BBM1)were used to characterize HER2(EQ)28 and HER2(EQ)BBξ. FIG. 4A depictsthe HER2 expression level of each of these lines. Flow cytometry (gatedon CAR+ T cells) was used to characterize CD107a degranulation and IFNyproduction in Mock (untransduced), HER2(EQ)28ξ or HER2(EQ)BBξ CAR Tcells following a 5 hr co-culture with either MDA-MB-361 tumor cells(low HER2 expressing) or BBM1 tumor cells (high HER2 expressing). Theresults of this analysis are presented in FIG. 4B. Similar studies wereconducted with the other breast cancer cells lines, and the results aresummarized in FIG. 4C. Production of IFNy production by HER2-CAR T cellsfollowing a 24 hr culture with recombinant HER2 protein or tumor targetswas measured by ELISA and the results of this analysis are shown in FIG.4D.

Example 7: In Vitro Anti-Tumor Activity

Flow cytometry was used to assess tumor cell killing following a 72hco-culture of Mock (untransduced), HER2(EQ)28ξ or HER2(EQ)BBξ CAR Tcells with tumor targets. The results of this analysis are presented inFIG. 5A. PD-1 and LAG-3 induction in total CAR T cells after a 72hco-culture with HER2-negative MDA-MB-468 or HER2-positive BBM1 cells wasmeasured, and the results of this analysis are presented in FIG. 5B.PD-1 induction in CD8+ CAR T cells following a 72h co-culture with tumortargets that are HER2-negative (LCL lymphoma, MDA-MB-468, U87 glioma),low-HER2 expressing (MDA-MB-361, 231BR) or high-HER2 expressing (SKBR3,BT474, BBM 1) was measured, and the results of this analysis arepresented in FIG. 5C. These studies suggest that HER2(EQ)BBξ causeslower PD-1 induction that does HER2(EQ)28ξ. Tumor cell killing withEffector:Tumor (E:T) ratio ranging from 0.25:1 to 2:1 was measured forboth HER2(EQ)28ξ or HER2(EQ)BBξ CAR T cells. The results of thisanalysis are presented in FIG. 5D, which shows that both HER2(EQ)28ξ andHER2(EQ)BBξ are effective in tumor cell killing in vitro. CFSEproliferation of HER2-CAR T cells following a 72h co-culture withMDA-MB-468 or BBMI cells was measured by flow cytometry. The results ofthis analysis are presented in FIG. 5E, which shows that HER2(EQ)BBξ CART cells proliferate more than HER2(EQ)28ξ CAR T cells.

Example 8: In Vivo Anti-Tumor Activity

The activity of intratumorally delivered HER2 CAR T cells was assessedin a patient-derived breast-to-brain metastasis model. FIGS. 6A-6C areH&E staining of tumors. Mice were treating by injection directly intothe tumor with Mock (untransduced) or HER2(EQ)BBξ CAR T cells. FIGS.6D-6F depict the results of optical imaging of the tumors and FIGS.6G-61 are Kaplan-Meier survival curves for mice treated locally witheither at day 3, 8 or 14 post tumor injection. These studies show thatHER2(EQ)BBξ CAR T cells have potent anti-tumor efficacy in vivo wheninjected directly into the tumor.

To assess anti-tumor efficacy in human xenograft models ofbreast-to-brain metastasis, BBM1 cells (0.2M) or BT474 (0.15M) wereintracranially injected in NSG mice. At day 8 post tumor injection,HER2(EQ)28ξ or HER2(EQ)BBξ, or Mock (untransduced) T cells (IM) wereinjected intratumorally. BBMI (FIG. 7A) and BT474 (FIG. 7B) tumors weremonitored by luciferase-based optical imaging. Kaplan Meier curves arepresented in FIG. 7C and FIG. 7D.

A human patient-derived orthotopic xenograft model of breast-to-brainmetastasis was also used to assess HER2(EQ)28ξ and HER2(EQ)BBξ CAR Tcells. FIG. 8A illustrates the region of tumor implantation bystereotactic injection of BBM1 cells (0.2M), and intraventricular T celldelivery. Staining of tumors is depicted in FIG. 8B. At day 14 posttumor injection, HER2(EQ)28ξ, HER2(EQ)BBξ, or Mock (untransduced) Tcells (0.5M) were injected intratumorally. Tumor growth was monitored byluciferase-based optical imaging. FIG. 8C presents the flux averages foreach treatment group, and FIG. 8D presents the Kaplan Meier survivalcurve for each treatment group.

Example 9: Additional CAR Targeted to HER2

FIGS. 9-14 depict the amino acid sequences of a various CAR havingdifferent linkers. Specifically, the CAR differ in the sequence andlength of the portion between the HER2 targeted scFv and thetransmembrane domain. The transmembrane domain is CD8, CD28 or CD28gg.The co-stimulatory domain is 4-1BB or CD28. All have a CD3ζ stimulatorydomain. In each case a T2A skip sequence separates the CAR from atruncated CD19 (CD19t) protein employed for cell tracking.

FIG. 15A schematically depicts various HER2 CAR that are identicalexcept for the sequence and length of the portion between the HER2 scFvand the CD8 transmembrane domain. All include a 4-1BB co-stimulatorydomain followed by a CD3ζ stimulatory domain. FIG. 15B depicts theresults of assays for CD19 and Protein L expression in T_(CM)transfected with a lentiviral vector expressing the indicated CAR. Ascan be seen from these results, transfection efficiency as assessed byCD19 expression was similar for both CAR. However, Protein L expressionwas lower for HER2(EQ)BBξ than for HER2(EQ)BBζ suggesting that theHER2(EQ)BBζ CAR is less stable that the HER2(EQ)BBζ. Analysis of cellexpansion (FIG. 15C) shows that none of the CAR interfere with T cellexpansion. FIGS. 16-18 show the results of additional studies showingthat a CAR with a very short spacer (FIG. 14) is relatively selectivefor CAR expressing high levels of HER2. Such CAR may be useful intreating HER2 expressing cancers where it is desirable to spare cellsexpressing a lower level of HER2 than the cancerous cells.

What is claimed is:
 1. A nucleic acid molecule encoding a chimericantigen receptor, wherein the chimeric antigen receptor comprises theamino acid sequence of any of SEQ ID NOs: 34-41.
 2. An isolatedpopulation of human T cells transduced by a vector comprising anexpression cassette encoding a chimeric antigen receptor, wherein thechimeric antigen receptor comprises the amino acid sequence of any ofSEQ ID NOs: 34-41.
 3. A method of treating a HER2 expressing braincancer in a patient comprising administering a population of autologousor allogeneic human T cells transduced by a vector comprising anexpression cassette encoding a chimeric antigen receptor, whereinchimeric antigen receptor comprises an amino acid sequence selected fromSEQ ID NOs:34-41.
 4. The method of claim 3 wherein the population ofhuman T cells comprise CD62L+ memory T cells.
 5. The method claim 3wherein the cancer is a breast to brain metastasis.
 6. The method ofclaim 3, wherein the transduced human T cells where prepared by a methodcomprising obtaining T cells from the patient, treating the T cells toisolate central memory T cells, and transducing at least a portion ofthe central memory cells to with a viral vector comprising an expressioncassette encoding a chimeric antigen receptor, wherein chimeric antigenreceptor comprises an amino acid sequence selected from SEQ IDNOs:34-41.
 7. The method of claim 3 wherein the T cells are administeredintratumorally.
 8. The method of claim 3 wherein the T cells areadministered intraventricularly.
 9. The method of claim 3 wherein the Tcells are administered intravetricularly adjacent to a tumor.
 10. Anisolated T cell expressing a polypeptide or a chimeric antigen receptorcomprising an amino acid sequence that is identical to an amino acidsequence selected from SEQ ID NOs:26-41.
 11. The nucleic acid moleculeof claim 1, wherein the chimeric antigen receptor comprises the aminoacid sequence of SEQ ID NO:
 34. 12. The nucleic acid molecule of claim1, wherein the chimeric antigen receptor comprises the amino acidsequence of SEQ ID NO:
 35. 13. The nucleic acid molecule of claim 1,wherein the chimeric antigen receptor comprises the amino acid sequenceof SEQ ID NO:
 36. 14. The nucleic acid molecule of claim 1, wherein thechimeric antigen receptor comprises the amino acid sequence of SEQ IDNO:
 37. 15. The nucleic acid molecule of claim 1, wherein the chimericantigen receptor comprises the amino acid sequence of SEQ ID NO:
 38. 16.The nucleic acid molecule of claim 1, wherein the chimeric antigenreceptor comprises the amino acid sequence of SEQ ID NO:
 39. 17. Thenucleic acid molecule of claim 1, wherein the chimeric antigen receptorcomprises the amino acid sequence of SEQ ID NO:
 40. 18. The nucleic acidmolecule of claim 1, wherein the chimeric antigen receptor comprises theamino acid sequence of SEQ ID NO:
 41. 19. A nucleic acid moleculeencoding a polypeptide, wherein the polypeptide comprises the amino acidsequence of any of SEQ ID NOs: 26-33.
 20. The nucleic acid molecule ofclaim 19, wherein the polypeptide comprises the amino acid sequence ofSEQ ID NO:
 26. 21. The nucleic acid molecule of claim 19, wherein thepolypeptide comprises the amino acid sequence of SEQ ID NO:
 27. 22. Thenucleic acid molecule of claim 19, wherein the polypeptide comprises theamino acid sequence of SEQ ID NO:
 28. 23. The nucleic acid molecule ofclaim 19, wherein the polypeptide comprises the amino acid sequence ofSEQ ID NO:
 29. 24. The nucleic acid molecule of claim 19, wherein thepolypeptide comprises the amino acid sequence of SEQ ID NO:
 30. 25. Thenucleic acid molecule of claim 19, wherein the polypeptide comprises theamino acid sequence of SEQ ID NO:
 31. 26. The nucleic acid molecule ofclaim 19, wherein the polypeptide comprises the amino acid sequence ofSEQ ID NO:
 32. 27. The nucleic acid molecule of claim 19, wherein thepolypeptide comprises the amino acid sequence of SEQ ID NO:
 33. 28. Anisolated population of human T cells transduced by a vector comprisingan expression cassette encoding a polypeptide, wherein the polypeptidecomprises the amino acid sequence of any of SEQ ID NOs: 26-33.
 29. Theisolated population of human T cells of claim 28, wherein thepolypeptide comprises the amino acid sequence of SEQ ID NO:
 26. 30. Theisolated population of human T cells of claim 28, wherein thepolypeptide comprises the amino acid sequence of SEQ ID NO:
 27. 31. Theisolated population of human T cells of claim 28, wherein thepolypeptide comprises the amino acid sequence of SEQ ID NO:
 28. 32. Theisolated population of human T cells of claim 28, wherein thepolypeptide comprises the amino acid sequence of SEQ ID NO:
 29. 33. Theisolated population of human T cells of claim 28, wherein thepolypeptide comprises the amino acid sequence of SEQ ID NO:
 30. 34. Theisolated population of human T cells of claim 28, wherein thepolypeptide comprises the amino acid sequence of SEQ ID NO:
 31. 35. Theisolated population of human T cells of claim 28, wherein thepolypeptide comprises the amino acid sequence of SEQ ID NO:
 32. 36. Theisolated population of human T cells of claim 28, wherein thepolypeptide comprises the amino acid sequence of SEQ ID NO:
 33. 37. Theisolated population of human T cells of claim 18, wherein the chimericantigen receptor comprises the amino acid sequence of SEQ ID NO:
 34. 38.The isolated population of human T cells of claim 18, wherein thechimeric antigen receptor comprises the amino acid sequence of SEQ IDNO:
 35. 39. The isolated population of human T cells of claim 18,wherein the chimeric antigen receptor comprises the amino acid sequenceof SEQ ID NO:
 36. 40. The isolated population of human T cells of claim18, wherein the chimeric antigen receptor comprises the amino acidsequence of SEQ ID NO:
 37. 41. The isolated population of human T cellsof claim 18, wherein the chimeric antigen receptor comprises the aminoacid sequence of SEQ ID NO:
 38. 42. The isolated population of human Tcells of claim 18, wherein the chimeric antigen receptor comprises theamino acid sequence of SEQ ID NO:
 39. 43. The isolated population ofhuman T cells of claim 18, wherein the chimeric antigen receptorcomprises the amino acid sequence of SEQ ID NO:
 40. 44. The isolatedpopulation of human T cells of claim 18, wherein the chimeric antigenreceptor comprises the amino acid sequence of SEQ ID NO:
 41. 45. Theisolated T cell of claim 10, wherein the polypeptide or chimeric antigenreceptor comprises an amino acid sequence that is identical to SEQ IDNO:26.
 46. The isolated T cell of claim 10, wherein the polypeptide orchimeric antigen receptor comprises an amino acid sequence that isidentical to SEQ ID NO:27.
 47. The isolated T cell of claim 10, whereinthe polypeptide or chimeric antigen receptor comprises an amino acidsequence that is identical to SEQ ID NO:
 28. 48. The isolated T cell ofclaim 10, wherein the polypeptide or chimeric antigen receptor comprisesan amino acid sequence that is identical to SEQ ID NO:
 29. 49. Theisolated T cell of claim 10, wherein the polypeptide or chimeric antigenreceptor comprises an amino acid sequence that is identical to SEQ IDNO:
 30. 50. The isolated T cell of claim 10, wherein the polypeptide orchimeric antigen receptor comprises an amino acid sequence that isidentical to SEQ ID NO:
 31. 51. The isolated T cell of claim 10, whereinthe polypeptide or chimeric antigen receptor comprises an amino acidsequence that is identical to SEQ ID NO:
 32. 52. The isolated T cell ofclaim 10, wherein the polypeptide or chimeric antigen receptor comprisesan amino acid sequence that is identical to SEQ ID NO:
 33. 53. Theisolated T cell of claim 10, wherein the polypeptide or chimeric antigenreceptor comprises an amino acid sequence that is identical to SEQ IDNO:34.
 54. The isolated T cell of claim 10, wherein the polypeptide orchimeric antigen receptor comprises an amino acid sequence that isidentical to SEQ ID NO:35.
 55. The isolated T cell of claim 10, whereinthe polypeptide or chimeric antigen receptor comprises an amino acidsequence that is identical to SEQ ID NO:
 36. 56. The isolated T cell ofclaim 10, wherein the polypeptide or chimeric antigen receptor comprisesan amino acid sequence that is identical to SEQ ID NO:
 37. 57. Theisolated T cell of claim 10, wherein the polypeptide or chimeric antigenreceptor comprises an amino acid sequence that is identical to SEQ IDNO:
 38. 58. The isolated T cell of claim 10, wherein the polypeptide orchimeric antigen receptor comprises an amino acid sequence that isidentical to SEQ ID NO:
 39. 59. The isolated T cell of claim 10, whereinthe polypeptide or chimeric antigen receptor comprises an amino acidsequence that is identical to SEQ ID NO:
 40. 60. The isolated T cell ofclaim 10, wherein the polypeptide or chimeric antigen receptor comprisesan amino acid sequence that is identical to SEQ ID NO: 41.